Mechanisms affecting targeting of Moesin to specific domains of the plasma membrane

在动物发育过程中，协同细胞分化形成组织需要在质膜（PM）和底层细胞骨架中建立独特的结构域，它们共同构成细胞皮层。我的研究项目主要集中在这些结构域如何在不同的细胞系中建立。这个过程中的一个关键因素是Moesin (Moe)，它可逆地将PM与皮质肌动蛋白细胞骨架连接起来。Moe中的分子内相互作用在“活性”和“非活性”状态之间循环。当与PM结合时，Moe被认为是“活跃的”，这一过程依赖于磷酸化并与磷脂酰肌醇4,5-二磷酸（PIP2）结合。然而，在某些情况下，Moe可以在没有磷酸化或PIP2结合的情况下具有活性。我们的假设是，Moe与PM相关蛋白的调节相互作用以及棕榈酰化等翻译后修饰也有助于将Moe带入“活性”状态。我们的目标是发现这些事件是如何影响Moe募集到细胞皮层的，这是在动物发育过程中特定组织类型分化所需的介导细胞粘附和增殖所必需的。一个重要的功能考虑是在PM上的Moe活性与其在细胞连接和切割沟中的其他作用的分化。因此，我们将比较神经干细胞（NSCs）和上皮细胞，前者的PM组织只需要Moe，而上皮细胞则需要PM和Moe的细胞连接作用。目的1将确定棕榈酰化如何调节Moe活性。我们在Moe中发现了两个潜在的棕榈酰化位点。我们发现这些位点的突变阻止了Moe向PM的招募。我们将在Moe中预测的棕榈酰化位点引入靶向突变，以确定它们分别如何影响NSCs和发育中的脑和翅盘上皮细胞的PM募集。目的2将描述我们发现的几种与Moe相互作用的pm相关蛋白。我们将这些基因鉴定为影响翼盘上皮细胞中Moe依赖性粘附的基因的前向遗传筛选和NSCs中Moe结合蛋白的质谱筛选的一部分。本科生HQP将分别描述一个或多个优先候选物，以确定它们如何影响Moe向PM的募集以及NSCs、上皮细胞和培养细胞的细胞连接。他们还将研究这如何影响moe介导的细胞增殖和分化调节。他们将比较使用特异性标记和间接免疫荧光检测的靶向体细胞镶嵌分析对粘附和增殖的影响。这将通过共免疫沉淀来补充，直接探测这些辅助因子在PM时在Moe复合物形成中的包含动力学。我的项目将继续推进我们对像Moe这样的蛋白质在细胞皮层中的亚结构域组织的理解，以及这是如何协调果蝇细胞增殖和分化的。

Cooperative cell differentiation to form tissues during animal development requires establishment of unique domains in the plasma membrane (PM) and underlying cytoskeleton, which together comprise the cell cortex. My research program focuses on how these domains are established in different cell lineages. A key factor in this process is Moesin (Moe), which reversibly links the PM to the cortical actin cytoskeleton. Intramolecular interactions in Moe cycle it between `active' and `inactive' states. Moe is thought to be `active' when bound to the PM, a process dependent on phosphorylation and binding to phosphatidylinositol 4,5-bisphosphate (PIP2). However, in some cases Moe can be active in the absence of phosphorylation or PIP2 binding. Our hypothesis is that regulated interactions of Moe with PM associated proteins and post-translational modifications like palmitoylation also help drive Moe into an `active' state. Our goal is to discover how these events affect Moe recruitment to the cell cortex that is required for mediating cell adhesion and proliferation needed for differentiation of specific tissue types during animal development. An important functional consideration is the differentiation of Moe activity at the PM versus its other roles at the cell junction and cleavage furrow. Thus, we will compare neural stem cells (NSCs), where Moe is required for PM organization only to epithelial cells, where both PM and cell junction roles for Moe are needed. Objective 1 will identify how palmitoylation regulates Moe activity. We identified two potential palmitoylation sites within Moe. We found that mutating these sites blocks Moe recruitment to the PM. We will introduce targeted mutations in the predicted palmitoylation sites in Moe to determine how they affect PM recruitment in NSCs and epithelial cells of the developing brain and wing disc, respectively. Objective 2 will characterize several PM-associated proteins we identified that interact with Moe. We identified these as part of a forward-genetic screen for genes affecting Moe-dependent adhesion in wing disc epithelial cells and a mass spectrometry screen for Moe binding proteins in NSCs. Undergraduate HQP will each characterize one or more prioritized candidates to determine how they affect Moe recruitment to the PM and cell junctions in NSCs, epithelial, and cultured cells. They will also examine how this affects Moe-mediated regulation of cell proliferation and differentiation. They will compare the effect of targeted somatic mosaic analysis on adhesion and proliferation using specific markers and indirect immunofluorescent detection. This will be complemented by co-immunoprecipitation, directly probing dynamics of the inclusion of these cofactors in Moe complex formation at the PM. My program will continue to advance our understanding of the organization of sub-domains in the cell cortex by proteins like Moe and how this coordinates cell proliferation and differentiation in Drosophila.